Method for culture of specimen



Aprll 19, 1955 T. J. cARsKl 2,706,702

METHOD FOR CULTURE OF SPECIMEN Filed Feb. 23, 1949 I N VEN TOR:

United States Patent METHOD FOR CULTURE OF SPECIMEN Theodore J. Carski, Baltimore, Md., assignor to Baltimore Biological Laboratory, Baltimore, Md., a corporation of Maryland Application February 23, 1949, Serial No. 77,863

7 Claims. (Cl. 195-126) The present invention relates to means whereby specimens of materials vulnerable to atmospheric conditions may be afforded the proper environment immediately upon withdrawal from their source and in the absence of contact with detrimental substances. More particularly, the invention relates to devices adapted to receive and house specimens for microbiological examination; specifically, it is concerned with devices carrying culture media maintained in a predetermined atmosphere suitable for the growth and viability of bacteria.

It is an object of the invention to provide an atmosphere of predetermined CO2 content within a microbiological culture receptacle prior to the introduction of a specimen for microbiological examination whereby the proper percentage of CO2 by weight may be maintained within the receptacle upon introduction of the sample or attained within the receptacle subsequent to introduction of the sample merely by the controlled admission of a diluting gas. It is another object to provide a culture receptacle designed to receive a fresh specimen within the proper culture medium and proper atmosphere immediately upon withdrawal of the specimen from its source. Further objects will be evident from the following disclosure.

In general terms, the invention comprises a gas-impermeable receptacle containing a culture medium and an atmosphere of predetermined CO2 content, the gas pressure within the container being preferably but not necessarily below that of the atmosphere and the receptacle having a closure means adapted for intake valving action whereby microbiological specimens and gaseous fluids may be drawn in without loss of the CO2. A specimen for microbiological examination may be withdrawn from its source and introduced directly into the receptacle without exposure to air by means of gravity, a pump, or suction created when the intake valve means of a reduced pressure receptacle is actuated. Predetermined amounts of a selected gas such as air may be allowed to follow the specimen into the receptacle if higher pressure within the receptacle or a higher oxygen content is desired. The valve means preferably is of a type whereby the receptacle immediately can then be sealed again from contact with the outside atmosphere.

The invention will be understood by reference to the accompanying drawings in which:

Fig. 1 is a culture medium housed in a receptacle with an elastic plug self-sealing type intake valve;

Fig. 2 is an enlarged vertical section taken on line 2-2 of Fig. 1 through the closure assembly of the receptacle shown in Fig. 1;

Fig. 3 is a culture medium housed in an ampoule type receptacle with a sealed tip type intake valve;

Fig. 4 is a vertical section through a modification of the apparatus of Fig. 1, showing a culture receptacle having a petcock type valve;

Fig. 5 is an elevation of a portion of the apparatus of Fig. 1, showing a specimen being introduced into the receptacle; and

Fig. 6 is a vertical section through a modification of the closure assembly of Fig. 2.

Referring to the drawings, the device of Fig. 1 comprises a glass bottle 10 containing a microbiological culture medium 11. The bottle is provided with an ordinary opening in its neck, in which opening is disposed a gas-impermeable closure 12 adapted to provide valving action. The closure comprises a tight fitting plug or stopper, composed of an elastic substance, sealing the ice opening from the passage of fluid but adapted to be penetrated by a hollow needle for the introduction of a specimen for microbiological examination, and gases or fluids if desired, and self-sealing upon withdrawal of the needle.

The culture receptacle is put into use as shown in Fig. 5 by inserting the hypodermic needle 23 through the elastic stopper 12 and introducing the specimen through the needle. When the specimen has been drawn into the bottle, the needle is withdrawn, allowing the puncture to seal itself.

The closure 12 is covered by a screw cap 13, which serves to hold the closure in place and to preserve the sterility of the surface of the closure. The screw cap may be provided with a perforation as indicated by the reference numeral 14 of Fig. 6, by means of which a needle may be inserted into the receptacle without the necessity of removing the cap, and in this event it may be desirable to utilize a cap having an imperforate disk liner 15 to maintain the sterility of the upper surface of the closure plug and which can be torn otf prior to puncture of the plug by a hypodermic needle. On the other hand, the screw cap is merely a preferred component of the device and may be dispensed with entirely if so desired, although it generally will be necessary to employ some form of clamp to prevent the elastic closure from blowing when the receptacle is autoclaved.

Referring to Fig. 3, a modified form of receptacle comprises an ampoule 16 carrying a culture medium 11 and a predetermined amount of CO2. 17 is sealed, and the ampoule neck 18 is provided with a length of tight fitting rubber tubing 19. When a specimen is to be introduced into the ampoule, the rubber tube is disconnected from the ampoule and connected at one end with the source of the specimen and the specimen is allowed to fill the tube. The tube is then pinched off near its free end and the free end is replaced on the ampoule neck, being fitted in gas-tight relationship therewith. Breaking of the glass tip within the rubber tubing permits entry of the specimen without exposure to air, and after introduction of the desired amount of specimen, the tube may be clamped to prevent access to the ampoule of additional fluids.

Fig. 4 illustrates a further modification of a receptacle closure for the device of the invention. A receptacle 20 carrying a culture medium 11 and a predetermined atmosphere is provided with a neck 21 in which is fixed a petcock 22 made gas-tight. The neck 24 is adapted for connection with a tube or the like carrying the specimen to be investigated, and the petcock 22 may be opened or closed at will for the introduction of the specimen and of air or other fluids as desired.

While glass strong bottles ordinarily prove satisfactory, it will be understood that the invention contemplates the use of receptacles of any appropriate size and shape and which may be fabricated from glass, metal, plastic or any material non-detrimental to the substances to be housed and which is sufliciently strong to withstand the pressures created upon evacuation.

Likewise, the type of closure employed is of no particular significance in the respect that any closure will prove suitable so long as it provides a gas-tight seal for the receptacle, will withstand the pressures and temperatures involved, is not detrimental to the substance to be housed, and can be conveniently made to function for intake valving action substantially as described above.

One method for preparing a pretreated microbiological culture medium in accordance with the invention is described as follows:

A culture medium of the broth type, specifically 50 ml. of a trypticase soy broth was dispensed by means of an automatic filling machine into a sterile strong glass bottle of about ml. capacity. The bottle was of the type having a screw threaded neck adapted to take a threaded cap. A self-sealing rubber diaphragm type closure was pushed into the opening in the bottle neck so as to firmly seal the bottle. After the rubber plug was in place, it was punctured with a hypodermic needle, and the bottle was evacuated through the needle until the internal pressure dropped to a point at which the broth began to boil. The internal pressure was dependent therefore on the temperature of the broth, e. g. it was The ampoule tip equal to the vapor pressure of the broth at the prevailing temperature. The internal pressure was about mm. of mercury since the broth was approximately at room temperature (27 C.).

When boiling of the broth had commenced, evacuation was discontinued and CO2 introduced into the evacuated bottle by the operation of a valve which closed off the vacuum pump system and opened a C02 system into the line. The CO2 system consisted of a device which measured a predetermined volume of CO2, and in this instance was made to deliver a quantity of CO2 equivalent to 3 ml. at atmospheric pressure and room temperature. An internal pressure rise of about 12 mm. mercury was observed as the CO2 was introduced, the final pressure within the bottle being about 35 mm. of mercury. The rubber closure was finally covered with a screw cap.

The bottle and its contents were next treated in an autoclave at a steam temperature of 121 C. for a 20- minute period for sterilizing, the screw cap eliminating danger of the closure blowing out. After cooling, the bottle was removed from the autoclave and held at incubating temperature (37 C.) for a period of about two weeks in order to test its sterility.

The pretreated culture medium was utilized for microbiological examination of a sample of human blood as follows:

Blood was withdrawn from a patient by means of a hypodermic needle in gas-tight connection on one end of a rubber tube to which a second needle or cannula was attached by a gas-tight fitting at the other end. Both the hypodermic needle and cannula were protected prior to use by sterile glass vial housings. When the tube became filled with blood, as evidenced by the flow of drops from the cannula at the other end of the tube, the flow was pinched oil and the cannula removed from its sterile glass-vial housing. The screw cap was then removed from the culture bottle and the cannula immediately forced through the rubber closure into the bottle. The fiow of blood through the rubber tubing was allowed to proceed until 15 ml. had been sucked into the bottle by the reduced pressure therein, the amount being measured by graduated indicia on the side of the bottle. At this point, the cannula was removed from the rubber closure, allowing the closure to seal itself, and the bottle was recapped.

Had the organism under investigation been of a type which required a higher percentage of oxygen, the appropriate conditions easily could have been reached by allowing a proper amount of air or other gas containing oxygen to be drawn in through the cannula immediately following introduction of the specimen of blood. The bottle finally was placed in storage at suitable incubation temperature for the promotion of growth of the organisms of interest.

While the invention finds its greatest utility in the investigation of specimens of microbes of the type which are stimulated by or require more carbon dioxide than is normally present in air, such as the genus Neisseria and the genus Brucella, it is evident that the invention also facilitates handling of other types, since it is a simple matter to provide the proper atmosphere in any case. Thus it is only necessary to admit air to the receptacle to obtain suitable atmospheric conditions for growth of meningococci, gonococci, Brucella abortus, and other fastidious pathogens, since although these are aerobic they do have a relatively high CO2 demand.

In regard to culture media suitable for use with the device of the invention, in addition to the trypticase soy broth mentioned above any type of medium, either liquid, solid, or semi-solid, which is normally employed for culture of organisms in a predetermined atmosphere may be utilized. Suitable liquid media include peptone broth, infusion broth, media for selective enrichment of organisms, media used for maintenance of viability, for storage, or for transportation of viable organisms, and various media for isolation, identification or culturing of organisms. Suitable solid and semi-solid media include peptone agar, infusion agar, differentially inhibiting or selective agars, and various media for isolation, identification or culturing of organisms. Of course, when media other than those liquids which will boil under reduced pressure at existing temperatures are employed, means other than observance of boiling must be used to determine when the required degree of receptacle evacuation has been reached.

The pressure resulting upon evacuation may suitably fall within the range of 10-750 mm. mercury in accordance with requirements. Obviously, the receptacles employed with high evacuation procedures must be sufficiently strong to withstand the pressure differentials created. Anaerobic organisms will in general require a very low oxygen tension but may tolerate relatively large amounts of CO2. On the other hand, certain aerobic organisms may require only a slight partial pressure of CO2, and in this event initial evacuation of the receptacle need be slight in order that a small volume of CO2 be introduced. If unusually large specimens are anticipated, it may be well to introduce even the small CO2 requirements into a high vacuum. If complete absence of oxygen is desired, it may be necessary to introduce nitrogen or hydrogen after evacuation, followed by the appropriate volume of CO2 necessary to attain a desirable final partial pressure of CO2.

In any event, the atmosphere within the receptacle is selected in accordance with requirements of the particular examination to be made. Ordinarily there will be a gas pressure within the receptacle as prepared for use of from 20 mm. to 760 mm. of mercury and the gas will consist essentially of CO2 but almost any possible ratio of CO2 and air may be required by certain organisms, and the amount of CO2 placed in the receptacle and the degree to which the receptacle is evacuated will vary accordingly, dependent upon the final conditions desired when the specimen has been introduced. If for some reason a final pressure of about 760 mm. is desired, it may be necessary to introduce the specimen under pressure or to allow escape of some of the gas within the receptacle when the specimen is introduced.

It will be understood that the concept of the present invention finds applicability whenever one substance vulnerable to normal atmospheres is to be brought into contact with a second substance and maintained in such contact with a suitable preselected atmosphere. That is to say, the invention in its broadest terms comprises means adapted to facilitate the immediate disposal of any kind of a specimen vulnerable to normal atmospheres within an environment conducive to the desired activity and viability of the organism.

I claim:

1. A method for immediately enhancing the viability of a microbiological organism contained in a specimen comprising the steps of establishing a substantially sterile isolated mass confined from contact with the atmosphere and consisting of a body of microbiological culture medium and a contiguous body of gas consisting essentially of carbon dioxide, introducing said specimen into said mass and into contact with said body of culture medium while excluding contact between said body of gas and the atmosphere, and maintaining said mass and said added specimen in an isolated and confined condition for a time sufiicient for growth of said organism contained in said specimen.

2. A method as defined in claim 1 wherein said body of gas is maintained at a pressure less than atmospheric pressure at least until the introduction of said specimen.

3. A method as defined in claim 1 wherein the introduction is accomplished substantially by reason of and under the influence of atmospheric pressure.

4. A method as defined in claim 1 and including the step of introducing a controlled amount of a selected gas into said mass subsequent to the introduction of said specimen and prior to the step of maintaining said mass and specimen.

5. A method for immediately enhancing the viability of a microbiological organism contained in a specimen comprising the steps of establishing a substantially sterile isolated mass confined from contact with the atmosphere and consisting of a body of microbiological culture medium substantially devoid of free oxygen and a contiguous body of carbon dioxide, admitting a measured amount of said specimen immediately subsequent to withdrawal of said specimen from its source into said mass and into contact with said body of culture medium while excluding contact between said body of carbon dioxide and the atmosphere, subsequently introducing a controlled amount of a selected gas into said mass and specimen thereby lowering the partial pressure of said body of carbon dioxide, and maintaining said mass, said added specimen and said amount of selected gas in an isolated and confined condition for a time sufficient for growth of said microbiological organism vulnerable to atmospheric conditions and contained in said specimen.

6. Method as defined in claim 5 including admitting said measured amount of said specimen under pressure.

7. Method as defined in claim 5 including establishing said contiguous body of carbon dioxide at a pressure less than atmospheric pressure and admitting said measured amount of said specimen under substantially atmos' pheric pressure.

References Cited in the file of this patent UNITED STATES PATENTS 1,137,388 Earp-Thomas Apr. 27, 1915 1,389,659 Hollowood Sept. 6, 1921 1,392,454 Sellman Oct. 4, 1921 1,413,703 Biehm Apr. 25, 1922 OTHER REFERENCES McCartney: Lancet, March 14, 1931, pages 585-586. McCartney: Lancet, August 19, 1933, pages 433-436. 10 Eimer and Amend, 85th Anniversary Catalogue, Laboratory Apparatus and Supplies, 1936, page 127.

Zinsser and Bayne-Jones, Textbook of Bacteriology, 7th ed., D. Appleton-Century Co., N. Y., pages 1084-1094. Schaub and Foley, Methods of Diagnostic Bacteriology, 15 1940, C. V. Mosby, St. Louis, pages 51, 53, 55, 57.

Gershenfeld, Bacteriology and Allied Subjects, 1945, Mack Pub. Co., Easton, Pa., pages 79 to 81. 

1. A METHOD FOR IMMEDIATELY ENHANCING THE VIABILITY OF A MICROBIOLOGICAL ORGANISM CONTAINED IN A SPECIMEN COMPRISING THE STEPS OF ESTABLISHING A SUBSTANTIALLY STERILE ISOLATED MASS CONFINED FROM CONTACT WITH THE ATMOSPHERE AND CONSISTING OF A BODY OF MICROBIOLOGICAL CULTURE MEDIUM AND A CONTIGUOUS BODY OF GAS CONSISTING ESSENTIALLY OF CARBON DIOXIDE, INTRODUCING SAID SPECIMEN INTO SAID MASS AND INTO CONTACT WITH SAID BODY OF CULTURE MEDIUM WHILE EXCLUDING CONTACT BETWEEN SAID BODY OF GAS AND THE ATMOSPHERE, AND MAINTAINING SAID MASS AND SAID ADDED SPECIMEN IN AN ISOLATED AND CONFINED CONDITION FOR A TIME SUFFICIENT FOR GROWTH OF SAID ORGANISM CONTAINED IN SAID SPECIMEN. 